Validated Reversed-Phase Ion-Interaction High-Performance Liquid Chromatography for Quantitation of Nitrate Content of (Willd.) Hook. f. ex Benth. Leaves.

This work sought to validate the reversed-phase ion-interaction high-performance liquid chromatography for quantifying the nitrate content in the extract and raw material of (Willd.) Hook. f. ex Benth. leaves. Three extraction methods (i.e., decoction, infusion, and ultrasound-assisted extraction) were investigated and compared. Furthermore, the effect of the solid-to-solvent ratio and defatting was also evaluated. The validation result showed that the high-performance liquid chromatographic method had a linear response (  = 0.9999) in the range of 1-50 g/mL. The limit of detection and limit of quantitation were 0.25 g/mL and 0.75 g/mL, respectively. In addition, the method was specific, precise, and accurate. So the validated method was suitable for determination of the nitrate content in leaves. Infusion of a nondefatted sample using a solid-to-solvent ratio of 1 : 10 gave the highest nitrate content in the raw material, 0.251 ± 0.003%. In case of a defatted sample, decoction provided the highest nitrate content, 0.309 ± 0.001%. Increasing the solid-to-solvent ratio and defatting had a huge effect on the nitrate content of leaves extracted from decoction. To the best of our knowledge, this is the first report that used the reversed-phase ion-interaction high-performance liquid chromatography for quantifying the nitrate content in leaves. Furthermore, the authors suggested that nitrate could be used as a standard marker for quality control of leaves' extract and raw material.